Human cytomegalovirus (HCMV) is a major cause of congenital birth defects as well as the cause of severe disease in immunocompromised adults. The pathology of HCMV diseases is related to the ability of the virus to infect many different cell types including epithelial and endothelial cells. The mechanisms by which the virus enters cells are poorly understood and represent the overall focus of the current proposal, as well as the long-term research interest of the applicant. Entry of HCMV requires the glycoprotein H/L (gH/gL), which is highly conserved among herpes viruses and directly mediates fusion between viral and cellular membranes during virus entry. Other proteins are added to gH/gL to form at least two distinct complexes, gH/gL/gO and gH/gl/UL128-131. It is clear that gH/gl/UL128-131 facilitates entry into epithelial and endothelial cells, likely via the binding of cell-type specific receptors. Due to its association with gH/gL, gO is assumed to also facilitate entry mechanisms, but evidence for this is lacking. Most previous studies of gO have been performed using laboratory-adapted strains of HCMV that are known to harbor extensive genetic mutations and rearrangement. Preliminary data are presented suggesting that gO is not a component of the wild type HCMV envelope. These data argue against a direct function in entry processes and suggest alternative roles for the interaction of gO with gH/gL. Aim 1 proposes a biochemical characterization of wild type gH/gL/gO using adenovirus vectors as well as mutants derived from wild type HCMV. Aim II involves mutagenesis of the UL131 protein to identify mutants that fail at different stages of the entry pathway. These mutants will allow for careful analysis of the cell type dependent entry mechanisms of HCMV.